In vitro models that recapitulate key aspects of native tissue architecture and the physical microenvironment are emerging systems for modeling development and disease. For example, the myocardium consists of layers of aligned and coupled cardiac myocytes that are interspersed with supporting cells and embedded in a compliant extracellular matrix (ECM). These cell-cell and cell-matrix interactions are known to be important regulators of tissue physiology and pathophysiology. In this protocol, we describe a method for mimicking the alignment, cell-cell interactions, and rigidity of the myocardium by engineering an array of square, aligned cardiac microtissues on polyacrylamide hydrogels. This entails three key methods (1) fabricating elastomer stamps with a microtissue pattern; (2) preparing polyacrylamide hydrogel culture substrates with tunable elastic moduli; and (3) transferring ECM proteins onto the surface of the hydrogels using microcontact printing. These hydrogels can then be seeded with cardiac myocytes or mixtures of cardiac myocytes and fibroblasts to adjust cell-cell interactions. Overall, this approach is advantageous because shape-controlled microtissues encompass both cell-cell and cell-matrix adhesions in a form factor that is relatively reproducible and scalable. Furthermore, polyacrylamide hydrogels are compatible with the traction force microscopy assay for quantifying contractility, a critical function of the myocardium. Although cardiac microtissues are the example presented in this protocol, the techniques are relatively versatile and could have many applications in modeling other tissue systems.Development of multicellular organisms depends on the proper establishment of signaling information in space and time. Secreted molecules called morphogens form concentration gradients in space and provide positional information to differentiating cells within the organism. Although the key molecular components of morphogen pathways have been identified, how the architectures and key parameters of morphogen pathways control the properties of signaling gradients, such as their size, speed, and robustness to perturbations, remains challenging to study in developing embryos. https://www.selleckchem.com/products/lb-100.html Reconstituting morphogen gradients in cell culture provides an alternative approach to address this question. Here we describe the methodology for reconstituting Sonic Hedgehog (SHH) signaling gradients in mouse fibroblast cells. The protocol includes the design of morphogen sending and receiving cell lines, the setup of radial and linear gradients, the quantitative time-lapse imaging, and the data analysis. Similar approaches could potentially be applied to other cell-cell communication pathways.Lineage-tracing experiments aim to identify and track the progeny and/or fate of cells. The use of inducible recombinases and fluorescent reporters has been instrumental in defining cellular hierarchies and allowing for the identification of stem cells in an unperturbed in vivo setting. The refinement of these approaches, labeling single cells, and the subsequent quantitative analysis of the clonal dynamics have allowed the comparison of different stem cell populations as well as establishing different mechanisms of cellular replenishment during steady-state homeostasis as well as during morphogenesis and disease. Utilizing this approach, it is now possible to establish the cellular hierarchy in a given tissue and the frequency of cell fate decisions on a population basis, thus providing a comprehensive analysis of cellular behavior in vivo. Although in this chapter we describe a protocol for lineage tracing of cells from fetal intestinal epithelium to the adult intestine, this approach can be widely applied to quantitatively assess the cell fate of any fetal cell during morphogenesis.Cell-fate determination is a function of cell-intrinsic and -extrinsic signaling cues. Understanding the design principles governing fate control in multicellular systems remains difficult to understand and analyze. To address the current challenges of spatial analysis of potential signaling events, we have developed a pipeline for assessment of the neighboring cells at defined areas in the vicinity of target cells using a newly defined concept of Neighborhood Impact Factor. We have used our pipeline to interrogate cellular decision-making in a genetically derived multi-lineage liver organoid from induced pluripotent stem cells. We examined endothelial versus hepatocyte fate determination for cells with similar expression level of an engineered driver gene circuit. Our analysis suggests that the relative level of gene expression to the neighbor population can control the final fate choice in our engineered liver multicellular system.Laboratory automation now commonly allows high-throughput sample preparation, culturing, and acquisition of microscopy images, but quantitative image analysis is often still a painstaking and subjective process. This is a problem especially significant for work on programmed morphogenesis, where the spatial organization of cells and cell types is of paramount importance. To address the challenges of quantitative analysis for such experiments, we have developed TASBE Image Analytics, a software pipeline for automatically segmenting collections of cells using the fluorescence channels of microscopy images. With TASBE Image Analytics, collections of cells can be grouped into spatially disjoint segments, the movement or development of these segments tracked over time, and rich statistical data output in a standardized format for analysis. Processing is readily configurable, rapid, and produces results that closely match hand annotation by humans for all but the smallest and dimmest segments. TASBE Image Analytics can thus provide the analysis necessary to complete the design-build-test-learn cycle for high-throughput experiments in programmed morphogenesis, as validated by our application of this pipeline to process experiments on shape formation with engineered CHO and HEK293 cells.The association of stereotypies and tics is not rare in children with severe autism spectrum disorder (ASD). The differential diagnosis between stereotypies and tics in this patient population can be difficult; however, it could be clinically relevant because of treatment implications.
A total of 108 video recordings of repetitive behaviors in young patients with stereotypies in the context of ASD were reviewed by a movement disorders expert and a trainee, in order to assess the prevalence of possible co-morbid tics. The Modified Rush Videotape Rating Scale (MRVS) was used to rate tic frequency and severity.
Out of 27 patients with stereotypies (24 males; mean age 14years), 18 (67%) reported possible tics. The most frequently observed tics were eye blinking, shoulder shrugging, neck bending, staring, and throat clearing. The mean MRVS score was 5, indicating mild tic severity. The only significant difference between patients with tics and patients without tics was the total number of stereotypies, which was higher in the subgroup of patients without tics (p?=?0.